Tushar Baran Deb

Multifunctional activities of human fibroblast 34-kDa hyaluronic acid-binding protein.

We have already reported that human fibroblast 34-kDa hyaluronic acid-binding protein (HABP) is identical with P32, the protein co-purified with splicing factor SF-2 [Deb and Datta (1996) J. Biol. Chem. 271, 2206-2212]. Data search further revealed that it has 92% sequence homology with a murine protein YL2 which interacts with HIV1 Rev. In this paper we have successfully demonstrated that HIV1 Rev binds with labeled 34-kDa HABP which can be competed with excess unlabeled HABP, suggesting this protein can be a cellular factor promoting HIV1 Rev to function. Interestingly, the multifunctional nature of HABP has been elucidated as it has 100% homology with another protein gC1q, the complement protein. The distinct non-overlapping binding motifs for HA and gC1q have been identified in the same protein, suggesting that either the protein can function independently or its activity is regulated by ligand binding, wherein its binding to one of the ligands may modulate the receptor activity of the other ligand.

Hyaluronic acid induced hyaluronic acid binding protein phosphorylation and inositol triphosphate formation in lymphocytes

In this report, the role of 34 kDa HA‐binding protein in hyaluronic acid‐induced cellular signalling in lymphocytes has been examined. The binding of 125I‐HA to lymphocytes in vivo was found to be inhibited by pre‐incubation of the cells with anti‐34 kDa HA‐binding protein antibodies, thus confirming 34 kDa HA‐binding protein as the specific HA‐receptor in lymphocytes. This observation was substantiated by anti‐34 kDa HA‐binding protein antibodies immunoblotting and 125I‐HA ligand blotting of lymphocytes cell lysate. The HA‐induced cell aggregation, tyrosine phosphorylation and cytoskeletal protein phosphorylation demonstrate the HA‐induced early cellular signalling events in lymphocytes. Further, to study the involvement of 34 kDa HA‐binding protein in mitogen induced lymphocyte signalling, we studied in vivo phosphorylation and secondary messenger formation. The enhanced 34 kDa HA‐binding protein phosphorylation by HA and the inhibition of cellular aggregation and IP3 formation by anti‐HA‐binding protein antibodies revealed that 34 kDa HA‐binding protein is one of the potential mediators in HA‐induced signal transduction.

Pnck induces ligand-independent EGFR degradation by probable perturbation of the Hsp90 chaperone complex

We have recently described a novel role for pregnancy-upregulated non-ubiquitous calmodulin kinase (Pnck) in the induction of ligand-independent epidermal growth factor receptor (EGFR) degradation (Deb TB, Coticchia CM, Barndt R, Zuo H, Dickson RB, and Johnson MD. Am J Physiol Cell Physiol 295: C365-C377, 2008). In the current communication, we explore the probable mechanism by which Pnck induces ligand-independent EGFR degradation. Pnck-induced EGFR degradation is calcium/calmodulin independent and is regulated by cell density, with the highest EGFR degradation observed at low cell density. Pnck is a novel heat shock protein 90 (Hsp90) client protein that can be co-immunoprecipitated with Hsp90. Treatment of Pnck-overexpressing cells with the pharmacologic Hsp90 inhibitor geldanamycin results in enhanced EGFR degradation, and destruction of Pnck. In cells in which Pnck is inducing EGFR degradation, we observed that Hsp90 exhibits reduced electrophoretic mobility, and through mass spectrometric analysis of immunopurified Hsp90 protein we demonstrated enhanced phosphorylation at threonine 89 and 616 (in both Hsp90-α and -β) and serine 391 (in Hsp90-α). Kinase-active Pnck protein is degraded by the proteasome, concurrent with EGFR degradation. A Pnck mutant (T171A) protein with suppressed kinase activity induced EGFR degradation to essentially the same level as wild-type (WT) Pnck, suggesting that Pnck kinase activity is not required for the induction of EGFR degradation. Although EGFR is degraded, overexpression of WT Pnck paradoxically promoted cellular proliferation, whereas cells expressing mutant Pnck (T171A) were growth inhibited. WT Pnck promoted S to G(2) transition, but cells expressing the mutant exhibited higher residency time in S phase. Basal MAP kinase activity was inhibited by WT Pnck but not by mutant T171A Pnck protein. Cyclin-dependent kinase (Cdk) inhibitor p21/Cip-1/Waf-1 was transcriptionally suppressed downstream to MAP kinase inhibition by WT Pnck, but not the mutant protein. Collectively, these data suggest that 1) Pnck induces ligand-independent EGFR degradation most likely through perturbation of Hsp90 chaperone activity due to Hsp90 phosphorylation, 2) EGFR degradation is coupled to proteasomal degradation of Pnck, and 3) modulation of basal MAP kinase activity, p21/Cip-1/Waf-1 expression, and cellular growth by Pnck is independent of Pnck-induced ligand-independent EGFR degradation.

Pregnancy-upregulated nonubiquitous calmodulin kinase induces ligand-independent EGFR degradation.

We describe here an important function of the novel calmodulin kinase I isoform, pregnancy-upregulated nonubiquitous calmodulin kinase (Pnck). Pnck (also known as CaM kinase Ibeta(2)) was previously shown to be differentially overexpressed in a subset of human primary breast cancers, compared with benign mammary epithelial tissue. In addition, during late pregnancy, Pnck mRNA was shown to be strongly upregulated in epithelial cells of the mouse mammary gland exhibiting decreased proliferation and terminal differentiation. Pnck mRNA is also significantly upregulated in confluent and serum-starved cells, compared with actively growing proliferating cells (Gardner HP, Seung HI, Reynolds C, Chodosh LA. Cancer Res 60: 5571-5577, 2000). Despite these suggestive data, the true physiological role(s) of, or the signaling mechanism(s) regulated by Pnck, remain unknown. We now report that epidermal growth factor receptor (EGFR) levels are significantly downregulated in a ligand-independent manner in human embryonic kidney-293 (HEK-293) cells overexpressing Pnck. MAP kinase activation was strongly inhibited by EGFR downregulation in the Pnck-overexpressing cells. The EGFR downregulation was not the result of reduced transcription of the EGFR gene but from protea-lysosomal degradation of EGFR protein. Knockdown of endogenous Pnck mRNA levels by small interfering RNA transfection in human breast cancer cells resulted in upregulation of unliganded EGFR, consistent with the effects observed in the overexpression model of Pnck-mediated ligand-independent EGFR downregulation. Pnck thus emerges as a new component of the poorly understood mechanism of ligand-independent EGFR degradation, and it may represent an attractive therapeutic target in EGFR-regulated oncogenesis.

Evidence for the existence of hyaluronectin-binding proteins in the plasma membranes

This report documents for the first time the existence of specific binding proteins for hyaluronic acid binding protein (hyaluronectin) in the plasma membranes of normal and transformed cells. Firstly, we showed the specific binding of hyaluronic acid binding protein to the cell surface of normal rat heart fibroblasts (NRHF) by saturation and competition methods using 125I‐labeled hyaluronic acid binding protein and calculated the binding dissociation constant (0.43 × 10−13 M). In order to identify hyaluronectin‐binding protein on the cell surface, plasma membranes isolated from rat brain, liver and flbrosarcoma were separated by SDS‐PAGE and transferred to nitrocellulose paper by electroblotting. Incubation of the transferred membrane proteins with 125I‐labeled hyaluronectin in the presence of non‐ionic as well as ionic detergents revealed two prominent bands of approximate molecular mass of 37 kDa and 40 kDa in brain, liver and flbrosarcoma. The specificity of the binding [125I]hyaluronectin to 37‐kDa and 40‐kDa membrane proteins was further confirmed, as the intensity of the bands was reduced in the presence of a 20‐fold excess of unlabeled hyaluronectin. We discuss our observations on hyaluronectin‐binding membrane proteins in the context of hyaluronectin‐mediated cellular functions.

AN INSIGHT INTO CELLULAR SIGNALLING MEDIATED BY HYALURONAN BINDING PROTEIN (HABP1)

We have reported the characterization of a cell surface glycoprotein of 34 kDa on SDS-PAGE, having specific affinity for hyaluronan and this protein has been termed as HABP1 by GDB (Ac. No. 9786126). The role of HABP1 in cell adhesion and tumor invasion has also been confirmed. In continuation, the gene encoding hyaluronan-binding protein from human fibroblast was isolated and its localization on human chromosome 17p12-p13 has been reported. Sequence analysis shows the identity of HABP1 with other proteins P-32, a protein co-purifying with splicing factor SF2; and gC1qR, the receptor for the globular head of C1q, indicating its multifunctional nature. Several reports suggest differential localization of this protein in various cell types. To confirm its role in signalling, the enhanced phosphorylation of HABP1 is being reported in mitogen activated cells and in sperms induced with progesterone and Calyculin A, the acrosome reaction and capacitation inducers. Another interesting observation is oligomerization of HABP1, which enhances its affinity for hyaluronan, highlighting its regulatory role in hyaluronan mediated signalling. Hexasaccharide of hyaluronan is the minimum chain length required for interaction with HABP1. Attempts are being further made to address the intricate mechanism by which this protein is phosphorylated and to understand how HABP1 phosphorylation is related to the signalling pathway and if there occurs any nuclear translocation after phosphorylation.